PURPOSE: A functional protein is required for structure/function analysis of cone photoreceptor cGMP-phosphodiesterase alpha' subunit (PDEalpha'). The purpose of this study was to express enzymatically active PDEalpha'. METHODS: Three expression vectors were constructed for transient and stable expression of PDEalpha': pC57 (transient) was obtained by subcloning bovine PDEalpha' cDNA into the pCIS2 expression vector; pNC57 (stable) was constructed by inserting the neo gene controlled by the mouse phosphoglycerate kinase-1 gene promoter into the pC57 vector; and pFC57 (transient) was generated by fusing the sequence encoding the FLAG peptide to the 5' end of the coding region of PDEalpha' cDNA. The recombinant plasmid DNAs were introduced into HEK293, CHO, or Y79 retinoblastoma cells using the calcium phosphate-mediated transfection procedure or lipofectamin. Northern and western blot hybridizations were used for RNA and protein analysis, respectively. RESULTS: Northern blots of both HEK293- and CHO-transfected cells showed strong expression of a 3 kb transcript corresponding to PDEalpha'. cGMP-PDE activity measured in homogenates of transiently and stably transfected cells ranged between 1.5 and 2.2 nmol cGMP hydrolyzed/min x mg total protein, a level of PDE activity slightly greater than that previously reported for the individual rod-photoreceptor PDE subunits transiently-expressed in HEK293 cells. Western blots of these cell homogenates showed a low level of expressed PDEalpha'. Transfection of Y79 retinoblastoma cells, that have been shown to express rod and cone PDEs endogenously, with the construct containing cone PDEa' cDNA fused to the FLAG peptide resulted in a protein with no enzymatic activity. CONCLUSIONS: Our results demonstrate that both HEK293 and CHO cells are capable of expressing functionally active cone PDEalpha'. High level of mRNA transcription and relatively low protein synthesis efficiency indicates the presence of a post-transcriptional control mechanism regulating overall expression of PDEalpha' in HEK293 and CHO cells.
PURPOSE: A functional protein is required for structure/function analysis of cone photoreceptor cGMP-phosphodiesterase alpha' subunit (PDEalpha'). The purpose of this study was to express enzymatically active PDEalpha'. METHODS: Three expression vectors were constructed for transient and stable expression of PDEalpha': pC57 (transient) was obtained by subcloning bovine PDEalpha' cDNA into the pCIS2 expression vector; pNC57 (stable) was constructed by inserting the neo gene controlled by the mousephosphoglycerate kinase-1 gene promoter into the pC57 vector; and pFC57 (transient) was generated by fusing the sequence encoding the FLAG peptide to the 5' end of the coding region of PDEalpha' cDNA. The recombinant plasmid DNAs were introduced into HEK293, CHO, or Y79 retinoblastoma cells using the calcium phosphate-mediated transfection procedure or lipofectamin. Northern and western blot hybridizations were used for RNA and protein analysis, respectively. RESULTS: Northern blots of both HEK293- and CHO-transfected cells showed strong expression of a 3 kb transcript corresponding to PDEalpha'. cGMP-PDE activity measured in homogenates of transiently and stably transfected cells ranged between 1.5 and 2.2 nmol cGMP hydrolyzed/min x mg total protein, a level of PDE activity slightly greater than that previously reported for the individual rod-photoreceptor PDE subunits transiently-expressed in HEK293 cells. Western blots of these cell homogenates showed a low level of expressed PDEalpha'. Transfection of Y79 retinoblastoma cells, that have been shown to express rod and cone PDEs endogenously, with the construct containing cone PDEa' cDNA fused to the FLAG peptide resulted in a protein with no enzymatic activity. CONCLUSIONS: Our results demonstrate that both HEK293 and CHO cells are capable of expressing functionally active cone PDEalpha'. High level of mRNA transcription and relatively low protein synthesis efficiency indicates the presence of a post-transcriptional control mechanism regulating overall expression of PDEalpha' in HEK293 and CHO cells.
Authors: Eric H Souied; Silvia N M Reid; Natik I Piri; Leonid E Lerner; Steven Nusinowitz; Debora B Farber Journal: Exp Eye Res Date: 2008-04-29 Impact factor: 3.467